LI Zhengyuan, XU Lianlian, SUN Jianyun, et al. Construction and Bioinformatics Analysis of TRPV4 Prokaryotic Expression Purification System[J]. Science and Technology of Food Industry, 2022, 43(3): 137−144. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2021060280.
Citation: LI Zhengyuan, XU Lianlian, SUN Jianyun, et al. Construction and Bioinformatics Analysis of TRPV4 Prokaryotic Expression Purification System[J]. Science and Technology of Food Industry, 2022, 43(3): 137−144. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2021060280.

Construction and Bioinformatics Analysis of TRPV4 Prokaryotic Expression Purification System

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  • Received Date: July 01, 2021
  • Available Online: November 30, 2021
  • Objective: This study aimed to construct the prokaryotic expression and purification system of non-selective cation channel TRPV4 protein and to carry out bioinformatics analysis to provide technical support for the study of diseases related to the target protein and the screening of drug active ingredients. Methods: The human TRPV4 gene was cloned into pET-28a(+), pET-32a, pET-15b, pGEX-5X-1, pEX-4T and pGEX-6p-1, respectively, and the prokaryotic expression system of TRPV4 was constructed using two E.coli expression hosts, BL21 and Rossetta. The target protein was isolated and purified by glutathione affinity chromatography and nickel column affinity chromatography. The target protein was analyzed by bioinformatics technology to obtain its corresponding physical, chemical properties and structural parameters. Results: The prokaryotic expression system of GST-TRPV4 and GST-TRPV4-6his fusion proteins was constructed using pGEX-6p-1 vector and Rossetta. The GST-TRPV4 fusion protein was significantly expressed when the IPTG concentrationwas 0.6 mmol/L, and the GST-TRPV4-6his fusion protein was significantly expressed when the IPTG concentration was 0.4 mmol/L, and the expression temperature was 18 ℃. When GST-TRPV4-6his fusion protein was purified, the complete GST-TRPV4-6his fusion protein could be obtained by imidazole solution at 100 mmol/L or 200 mmol/L. Conclusion: In this paper, human TRPV4 ion channel protein was successfully constructed, expressed and purified in prokaryotic expression system for the first time, and the physicochemical properties of the protein were analyzed by bioinformatics, which laid a good foundation for subsequent high-purity mass expression and further study.
  • [1]
    GORETZKI B, GLOGOWSKI N A, DIEHL E, et al. Structural basis of TRPV4 N terminus interaction with syndapin/PACSIN1-3 and PIP2[J]. Structure,2018,26(12):1583−1593. doi: 10.1016/j.str.2018.08.002
    [2]
    TSUNO N, YUKIMASA A, YOSHIDA O, et al. Discovery of novel 2′, 4′-dimethyl-[4, 5′-bithiazol]-2-yl amino derivatives as orally bioavailable TRPV4 antagonists for the treatment of pain: Part 1[J]. Bioorganic & Medicinal Chemistry Letters,2016,26(20):4930−4935.
    [3]
    BRIAN G L, EDWARD J B, DAVID J B. Recent advances in TRPV4 agonists and antagonists[J]. Bioorganic & Medicinal Chemistry Letters,2020,30:127022.
    [4]
    MARTINAC B, POOLE K. Mechanically activated ion channels[J]. The International Journal of Biochemistry & Cell Biology,2018,97:104−107.
    [5]
    PAIRET N, MANG S, KIECHLE T, et al. Differential modulation of transendothelial electrical resistance by TRPV4 agonists is mediated by apoptosis and/or necrosis[J]. Biochemistry and Biophysics Reports,2019,20:100672. doi: 10.1016/j.bbrep.2019.100672
    [6]
    BAXTER M, ELTOM S, DEKKAK B, et al. Role of transient receptor potential and pannexin channels in cigarette smoke-triggered ATP release in the lung[J]. Thorax:The Journal of the British Thoracic Society,2014,69(12):1080−1089.
    [7]
    CENAC N, BAUTZOVA T, LE FAOUDER P, et al. Quantification and potential functions of endogenous agonists of transient receptor potential channels in patients with irritable bowel syndrome[J]. Gastroenterology,2015,149(2):433−444. doi: 10.1053/j.gastro.2015.04.011
    [8]
    YAN J J, YE F, YING J, et al. Cimifugin relieves pruritus in psoriasis by inhibiting TRPV4[J]. Cell Calcium,2021,97:102429. doi: 10.1016/j.ceca.2021.102429
    [9]
    FICHNA J, MOKROWIECKA A, CYGANKIEWICEZ A I, et al. Transient receptor potential vanilloid 4 blockade protects against experimental colitis in mice: A new strategy for inflammatory bowel diseases treatment?[J]. Neurogastroenterology & Motility the Official Journal of the European Gastrointestinal Motility Society,2012,24(11):e557−e560.
    [10]
    OLIVAN V A, GARCIA O A, LOZANO G J, et al. Pharmacological activation of TRPV4 produces immediate cell damage and induction of apoptosis in human melanoma cells and Ha Ca T keratinocytes[J]. PloS One,2018,13(1):e190307.
    [11]
    FIORIO P A, ONG H L, CHENG K T, et al. TRPV4 mediates tumor-derived endothelial cell migration via arachidonic acid-activated actin remodeling.[J]. Oncogene,2012,31:200−212. doi: 10.1038/onc.2011.231
    [12]
    ADAPALA R K, THOPPIL R J, GHOSH K, et al. Activation of mechanosensitive ion channel TRPV4 normalizes tumor vasculature and improves cancer therapy[J]. Oncogene,2016,35:314−322. doi: 10.1038/onc.2015.83
    [13]
    SHIGEMATSU H, SOKABE T, DANEV R, et al. A 3.5-nm structure of rat TRPV4 cation channel revealed by Zernike phase-contrast cryoelectron microscopy[J]. Journal of Biological Chemistry,2010,285(15):11210−11218. doi: 10.1074/jbc.M109.090712
    [14]
    ZHAN L, LI J. The role of TRPV4 in fibrosis[J]. Gene,2018,642:1−8. doi: 10.1016/j.gene.2017.10.067
    [15]
    WORTLEY M A, BIRRELL M A, BELVISI M G. Drugs affecting TRP channels[J]. Pharmacology and Therapeutics of Asthma and COPD,2016,63:213−241.
    [16]
    GARCIA E A, MRKONJIC S, JUNG C, et al. The TRPV4 channel[J]. Handb Exp Pharmacol,2014,222:293−319.
    [17]
    SURESH K, SERVINSKY L, REYES J, et al. Hydrogen peroxide-induced calcium influx in lung microvascular endothelial cells involves TRPV4[J]. Am J Physiol Lung Cell Mol Physiol,2015,309(12):L1467−L1477. doi: 10.1152/ajplung.00275.2015
    [18]
    SCHERAGA R G, SOUTHERN B D, GROVE L M, et al. The role of transient receptor potential vanilloid 4 in pulmonary inflammatory diseases[J]. Frontiers in Immunology,2017,8:503. doi: 10.3389/fimmu.2017.00503
    [19]
    LAMANDE S R, YUAN Y, GRESSHOFF I L, et al. Mutations in TRPV4 cause an inherited arthropathy of hands and feet[J]. Nature Genetics,2011,43(11):1142−1146. doi: 10.1038/ng.945
    [20]
    LIU Y Z, CHEN X, LI Z Y, et al. Ansavaricin J, a new heterocyclic ring-fused streptovaricin from gene stvP5-deleted mutant of Streptomyces spectabilis CCTCC M2017417[J]. Chemistry & Biodiversity,2020,17(8):e1900713.
    [21]
    LIU Y Z, CHEN X, LI Z Y, et al. Functional analysis of cytochrome P450s involved in streptovaricin biosynthesis and generation of anti-MRSA analogues[J]. ACS Chemical Biology,2017,12(10):2589−2597. doi: 10.1021/acschembio.7b00467
    [22]
    SHIBASAKI K. TRPV4 activation by thermal and mechanical stimuli in disease progression[J]. Laboratory Investigation,2020,100:218−223. doi: 10.1038/s41374-019-0362-2
    [23]
    HOSHI Y, OKABE K, SHIBASAKI K, et al. Ischemic brain injury leads to brain edema via hyperthermia-induced TRPV4 activation[J]. The Journal of Neuroscience,2018,38(25):5700−5709. doi: 10.1523/JNEUROSCI.2888-17.2018
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